Human immunodeficiency virus type 1 (HIV) is the causative agent of the acquired immunodeficiency syndrome, or ADS. WHO estimates that, by the end of this year, 0.7 percent of the world's population will be infected with HIV. Highly active anti-retroviral therapy (HAART) has improved the quality of life for many HIV seropositive individuals in the First World. Most other infected individuals have no access to HAART, and HAART is not always effective. Because a vaccine is not yet available, there are compelling reasons to develop novel therapeutics, including the introduction of gene-modified cells to protect against HIV. Vectors based upon murine leukemia virus (MLV) have been widely used as gene transfer vehicles. Unfortunately, efficient transduction by MLV requires mitosis. HIV-based vectors have demonstrated remarkable promise in their ability to transduce a variety of resting human cells, including CD34+ subpopulations that are thought to represent primitive hematopoietic stem cells (HSC). This proposal concentrates on HSC subset transduction and repopulation analyses and the development of HIV-based gene therapy vectors directed against HIV itself. Careful comparisons will be made between different HSC subsets (e.g., CD34+, lin-CD34-, CD34+KDR+, CD34+KDR-, and 'side-population' cells) in their ability to be transduced by HIV vectors. Readout will include methylcellulose. colony and LT-CIC assays, and repopulation of NOD/SCID mice by transduced cells. To minimize variability, the HSC subsets will be separately transduced with HIV vectors encoding different transgenes and engrafted in the same mouse. In parallel with those studies, HIV vectors encoding both CCR5 and CXCR4 intrakines will be constructed. An inducible vector employing the 'gene switch' system will be used for the SDF-KDEL intrakine since CXCR4 is critical for stem cell homing. These vectors will be initially used to transduce T cells and macrophages to evaluate protection against HTV infection. Once efficacy is demonstrated, the intrakine vectors will be used for HSC transduction and differentiation in the SCID-hu thy/liv model. Resultant transduced thymocytes will be challenged with H1V ex vivo to determine whether the intrakines have a protective effect. Lastly, a suicide HIV vector encoding HSV thymidine kinase will be constructed, inducible in the presence of both Tat and Rev. In the presence of ganciclovir, transduced cells which also express Tat and Rev will undergo cell death. This vector will be tested in established and primary T cells for efficacy of HIV inhibition. This will be compared to the intrakine-encoding vector above and an antisense envelope construct, both alone and in combination. These intervector comparisons should serve to identify the superior transgene combination. If successful, these studies may facilitate the development of clinical-grade anti-HIV lentiviral vectors for transduction of HSC.